Chemical Composition and Biological Activities of Gerbera anandria

Gerbera anandria (Compositae) was extracted with 75% ethanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. Column chromatography of the light petroleum fraction resulted in the isolation of methyl hexadecanoate, while the chloroform fraction afforded xanthotoxin, 2-hydroxy-6-methylbenzoic acid, 7-hydroxy-1(3H)-isobenzofuranone, a mixture of β-sitosterol and stigmasterol, and 8-methoxysmyrindiol and the ethyl acetate fraction gave gerberinside, apigenin-7-O-β-d-glucopyranoside and quercetin. A new coumarin, 8-methoxysmyrindiol, was found. The chemical structures of the isolated compounds were established by MS and NMR (HSQC, HMBC). Free radical scavenging and cytotoxic activities of crude extracts and 8-methoxysmyrindiol were further investigated. The ethyl acetate phase exerted the strongest DPPH free radical scavenging activity in comparison to the other fractions. The coumarin 8-methoxysmyrindiol demonstrated cytotoxicity against multiple human cancer cell lines, with the highest potency in HepG2 cells.


Introduction
Gerbera anandria is a plant from the Compositae family, a perennial herb of the Gerbera genus, a well-known traditional Chinese medicinal herb and widely grown in China. It is used in Chinese folk medicine to treat coughs, sore throats, as an effective anti-inflammatory, for detoxification, and expelling wind, and is mainly used for rheumatic pains, arthralgia, bruises, sprains, and dysentery. A review of the literature indicates that only a few studies have been conducted on Gerbera anandria. Coumarins have been isolated and detected, such as 4-hydroxy-5-methylcoumarin [1], gerberacoumarin [2] and daphnetin 7-methyl ether [3]. Two benzofurans with ethanoyl groups on the pyridine ring were isolated too [4]. Flavonoids such as quercetin were detected in the flowers using high-performance liquid chromatography (HPLC). Also, β-sitosterol and stigmasterol have been reported from this species [5,6]. Coumarins from Gerbera anandria were found to have broad spectrum anti-tumor and antibacterial activities in vitro and in vivo and are considered to be potential new cancer chemoprevention agents. This prompted us to isolate and investigate the chemical composition, as well as the free radical scavenging activity, and in vitro cytotoxic activities of the total extract, light petroleum, chloroform and ethyl acetate fractions and some compounds isolated from Gerbera anandria in order to evaluate its pharmacological potential.

Results and Discussion
Fractionation of the extracts yielded well-known substances, most of which, however, had not yet been described as constituents of G. anandria. Meanwhile a new coumarin, 8-methoxysmyrindiol, was found. The chemical structures of the isolated compounds ( Figure 1) were identified using mass spectrometry (MS, Figure 2) as well as nuclear magnetic resonance (1D-and 2D-NMR), including heteronuclear multiple quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC, Figure 3).

Antioxidant Activity
The DPPH assay is based on measuring the scavenging ability of antioxidants towards the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) [7]. The free radical DPPH is reduced to the corresponding hydrazine when it reacts with hydrogen donors [8]. The DPPH assay is considered a valid and easy assay to evaluate the antioxidant activity of natural products [9]. The IC 50 values of different fractions and the standard antioxidant are given in Table 1. The results display the ability of the ethyl acetate fraction to scavenge DPPH, with a stronger scavenging effect than the other fractions. Of the tested compounds, Ascorbic acid used as the positive control, exerted the strongest activity. Most of the isolated compounds from G. anandria showed lower IC 50 s than the crude extract.   Table 2 summarizes the IC 50 s of the chloroform fraction and 8-methoxysmyrindiol against various human cancer cell lines. Both show high IC 50 values in the chemotherapy resistant HepG2 cell line in comparison to the others. Cytotoxicity of chloroform fraction was more pronouncing against HeLa, A375-S2, HT1080 and HL60 cells than 8-methoxysmyrindiol. On the other hand, 8-methoxysmyrindiol was more cytotoxic against MCF-7, HCT11 and A549cells than the chloroform fraction.

Plant Material
Fresh Gerbera anandria plants were purchased in June 2011 from HeBei Anguo herbal medicine market and air dried. The plant material was botanically identified by Taiming Wei of Harbin Medical University, and a voucher specimen (NO2012809) is deposited in the Herbarium of the Department of Taxonomy, Harbin Medical University, Daqing, China.

Extraction Procedure
The whole plants of Gerbera anandria (10 kg) were exhaustively extracted with 75% aqueous ethanol (3 × 160 L × 2h) at 80 °C. The ethanolic extract was filtered and then concentrated under vacuum to yield 750 g of a viscous residue. The residue was suspended in water and successively partitioned against light petroleum (b.p. 60-90 °C), chloroform and ethyl acetate. The organic solvents were evaporated under vacuum using rotary evaporator at lower temperature to yield 90 g, 65 g and 60 g of final residues, respectively.

Compound 1
The light petroleum fraction (10 g) redissolved in chloroform was mixed with 10 g silica gel for column chromatography. The dry mixed initial zone was chromatographed on a silica gel column (120 × 2 cm, 200 g) at room temperature. The column was eluted with a gradient using a mixture of chloroform-methanol as mobile phase. Fractions of 200 mL each were collected and column fractions 123-126 eluted at a chloroform-methanol (CHCl 3 -CH 3 OH), (100:1, v/v) were collected, evaporated, concentrated under vacuum and monitored by TLC using a mixture of petroleum-acetone (2:1, v/v) as a system for TLC development. Spots were detected after spraying with H 2 SO 4 10% (v/v) in ethanol followed by heating at 105 °C for 5 min, and 15 mg of compound 1 was obtained as white oil from petroleum fraction.

Compounds 2-6
The chloroform fraction (65 g) redissolved in chloroform was mixed with 65 g silica gel for column chromatography and the dry mixed initial zone was chromatographed on a silica gel column (140 × 4 cm, 1 kg) at room temperature, the column was eluted with chloroform-methanol (100:2) as mobile phase, fractions of 5,000 mL were collected, concentrated under vacuum at lower temperature to yield 10 g of final residue. The residue was dissolved in chloroform again and the solution was mixed with 10 g silica gel for column chromatography and the dry mixed initial zone was chromatographed on a silica gel column (120 × 2 cm, 200 g) at room temperature. The column was eluted with a gradient using a mixture of dichloromethane-methanol as mobile phase, Fractions of 200 mL each were collected, evaporated, concentrated under vacuum and monitored by TLC using a mixture of petroleum-acetone (1:1, v/v) as a system for TLC development. Spots were detected after spraying with H 2 SO 4 10%(v/v) in water followed by heating at 105 °C for 5 min, Column fractions 43-176 eluted at a CHCl 2 -CH 3 OH (100:1, v/v) were collected, evaporated, fractions having the same R f were pooled and purified either by crystallization or by preparative TLC or by Sephadex LH-20 gel column chromatography,

Compounds 7-9
The ethyl acetate fraction (60 g) redissolved in chloroform was mixed with 60 g silica gel for column chromatography and the dry mixed initial zone was chromatographed on a silica gel column (140 × 4 cm, 1 kg) at room temperature, the column was eluted with chloroform-methanol (100:10) as mobile phase, fractions of 6,000 mL were collected, concentrated under vacuum at lower temperature to yield 8 g of final residue; Then the residue was dissolved in chloroform again and the solution was mixed with 8 g silica gel for column chromatography and the dry mixed initial zone was chromatographed on a silica gel column (120 × 2 cm, 160 g) at room temperature. The column was eluted with a gradient using a mixture of dichloromethane-methanol as mobile phase, Fractions of 200 mL each were collected, concentrated under vacuum and monitored by TLC using chloroformmethanol (5:2, v/v) as developing system. The TLC was visualised under UV and 3% AlCl 3 (v/v) in ethanol spray reagent. Column fractions 47-213 eluted at a CHCl 2 -CH 3 OH (100:5, v/v) were collected, evaporated and fractions having the same R f were pooled and purified either by crystallisation or by preparative TLC or by Sephadex LH-20 gel column chromatography to afford compounds 7-9. H-4,(δ6.98) with C-3, C-7a, C-6; H-6 (δ6.94 ) with C-7, C-4, C-7a, C-1; H2-3 (δ5.33) with C-3a, C-1, C-5, C-7a, C-4, C-6, C-7. Compound 4 was identified as 7-hydroxy-1(3H)-isobenzofuranone from these spectral data and physical properties [13].  -29). This compounds was identified as a mixture of β-sitosterol (5a) and stigmasterol (5b) from these spectral data and physical properties [14,15]. .50 (C-7''). Compound 9 was identified as apigenin-7-O-β-D-glucopyranoside from these spectral data and physical properties [18].

Antioxidant Activity
Stock solutions of the crude methanol extract and fractions (light petroleum, chloroform, ethyl acetate) were prepared by dissolving 10 mg in 1 mL methanol. Serial dilution was done from each stock solutions and 1mL from each dilution was tested individually by adding to 2 mL of 0.05 mg·mL −1 2,2-diphenyl-1-picrylhydrazyl (DPPH). The mixtures were vigorously shaken and allowed to stand in the dark for 15 min at room temperature. Absorbance was measured at 515 nm against methanol as blank. The inhibition rate of each extract against DPPH radical was calculated according to the following formula: The curve was drawn with the clearance Y as ordinate and the mass concentration m as abscissa. The free radical scavenging activity, expressed as IC 50 (μg·mL −1 ), was compared with standard antioxidants such as ascorbic acid.

Adherent Cells
Sensitivity of the cells to drugs was determined in triplicate using the MTT cell viability assay [19,20]. Exponentially growing adherent tumor cell lines (5 × 10 4 /mL ) were seeded in a 96-well plate (Corning, Corning, NY, USA) after trypsinization [21], 100 μL each hole. After incubation for 24 h the cells were incubated with 100 μL fresh medium containing various concentrations of compounds at 37 °C for 48 h. The medium was removed and cells were incubated with 100 μL fresh medium containing 0.5 mg·mL −1 MTT. During incubation for further 4 h, MTT is reduced by mitochondrial dehydrogenases of viable cells to a purple formazan product. The medium was discarded and 150 μL DMSO was added. The plates were shaken for 10 min at room temperature, and the spectrophotometric absorbance was monitored at 492 nm using Tecan-ELISA plate reader.

Suspension Cells
Exponentially growing cell lines (2 × 10 5 /mL)were seeded in a 96-well plate, 50 μL Each hole (Corning). After incubation for 24 h the cells were incubated with 50 μL fresh medium containing various concentrations of compounds at 37 °C for 48 h. The medium was removed and cells were incubated with 10 μL fresh medium containing 5 mg·mL −1 MTT. During incubation for further 4 h, MTT is reduced by mitochondrial dehydrogenases of viable cells to a purple formazan product. The medium was discarded and formazan crystal dissolved in 100 μL triple solution (SDS 10 g, 10M HCl 0.1 mL, isobutanol 5 mL, diluted with distilled water to 100 mL). After incubation for further 12 h at 37 °C, the spectrophotometric absorbance was monitored at 492 nm using Tecan-ELISA plate reader.

Evaluation of Results
All experiments were repeated at least three times. Results are reported as means ± SE. The IC50 was determined as the drug concentration which resulted in a 50% reduction in cell viability or inhibition of the biological activity. The inhibition rate (Y) was calculated according to the following formula: Y = (A 0 -A i )/A 0 × 100% A 0 : The absorbance of Negative control; A i : The absorbance of adding the sample solutions.

Conclusions
A new coumarin 8-methoxysmyrindiol and eight other compounds were isolated from the 75% aqueous ethanol extract of the whole plants of Gerbera anandria. The chemical structures of the isolated compounds were established by MS and NMR (APT, HSQC and HMBC). Free radical scavenging and cytotoxic activities of crude extracts and 8-methoxysmyrindiol were further investigated. The ethyl acetate phase exerted the strongest DPPH free radical scavenging activity in comparison to other fractions, this may be due to the fact that the majority of the flavonoids are normally present in the ethyl acetate phase. The coumarin 8-methoxysmyrindiol demonstrated cytotoxicity against multiple human cancer cell lines, with the highest potency in HepG2 cells.

Author Contributions
The listed authors contributed to this work as described in the following. Fa He, Miao Wang and Minghuan Gao carried out the extraction and isolation. Min Zhao and Fa He participated in the structural elucidation. Minghuan Gao and Fa He conducted the MTT colorimetric assay and helped interpreting the results. As corresponding author Yuhua Bai and Chunjie Zhao organized the study and participated in the structural elucidation. All authors helped preparing the manuscript and approved the final version.

Conflicts of Interest
All authors declare that there is no potential conflict of interest.